Non-canonical type 1 cannabinoid receptor signaling regulates night visual processing in the inner rat retina.

Type 1 cannabinoid receptors (CB1Rs) are expressed in major retinal neurons within the rod-pathway suggesting a role in regulating night visual processing, but the underlying mechanisms remain poorly understood. Using acute rat retinal slices, we show that CB1R activation reduces glutamate release from rod bipolar cell (RBC) axon terminals onto AII and A17 amacrine cells through a pathway that requires exchange proteins directly activated by cAMP (EPAC1/2) signaling. Consequently, CB1R activation abrogates reciprocal GABAergic feedback inhibition from A17 amacrine cells. Moreover, the activation of CB1Rs in vivo enhances and prolongs the time course of the dim-light rod-driven visual responses, an effect that was eliminated when both GABAA and GABAC receptors were blocked. Altogether, our findings underscore a non-canonical mechanism by which cannabinoid signaling regulates RBC dyad synapses in the inner retina to regulate dim-light visual responses to fine-tune night vision.

Autocrine activation of P2X7 receptors mediates catecholamine secretion in chromaffin cells.

BACKGROUND AND PURPOSE: ATP is highly accumulated in secretory vesicles and secreted upon exocytosis from neurons and endocrine cells. In adrenal chromaffin granules, intraluminal ATP reaches concentrations over 100 mM. However, how these large amounts of ATP contribute to exocytosis has not been investigated. EXPERIMENTAL APPROACH: Exocytotic events in bovine and mouse adrenal chromaffin cells were measured with single cell amperometry. Cytosolic Ca2+ measurements were carried out in Fluo-4 loaded cells. Submembrane Ca2+ was examined in PC12 cells transfected with a membrane-tethered Ca2+ indicator Lck-GCaMP3. ATP release was measured using the luciferin/luciferase assay. Knockdown of P2X7 receptors was induced with short interfering RNA (siRNA). Direct Ca2+ influx through this receptor was measured using a P2X7 receptor-GCamp6 construct. KEY RESULTS: ATP induced exocytosis in chromaffin cells, whereas the ectonucleotidase apyrase reduced the release events induced by the nicotinic agonist dimethylphenylpiperazinium (DMPP), high KCl, or ionomycin. The purinergic agonist BzATP also promoted a secretory response that was dependent on extracellular Ca2+. A740003, a P2X7 receptor antagonist, abolished secretory responses of these secretagogues. Exocytosis was also diminished in chromaffin cells when P2X7 receptors were silenced using siRNAs and in cells of P2X7 receptor knockout mice. In PC12 cells, DMPP induced ATP release, triggering Ca2+ influx through P2X7 receptors. Furthermore, BzATP, DMPP, and KCl allowed the formation of submembrane Ca2+ microdomains inhibited by A740003. CONCLUSION AND IMPLICATIONS: Autocrine activation of P2X7 receptors constitutes a crucial feedback system that amplifies the secretion of catecholamines in chromaffin cells by favouring submembrane Ca2+ microdomains.

Glucagon Increases Retinal Rod Bipolar Cell Inhibition Through a D1 Dopamine Receptor-Dependent Pathway That Is Altered After Lens-Defocus Treatment in Mice.

Purpose: Members of the secretin/glucagon family have diverse roles in retinal physiological and pathological conditions. Out of them, glucagon has been associated with eye growth regulation and image defocus signaling in the eye, both processes central in myopia induction. On the other hand, dopamine is perhaps the most studied molecule in myopia and has been proposed as fundamental in myopia pathogenesis. However, glucagonergic activity in the mammalian retina and its possible link with dopaminergic signaling remain unknown. Methods: To corroborate whether glucagon and dopamine participate together in the modulation of synaptic activity in the retina, inhibitory post-synaptic currents were measured in rod bipolar cells from retinal slices of wild type and negative lens-exposed mice, using whole cell patch-clamp recordings and selective pharmacology. Results: Glucagon produced an increase of inhibitory post-synaptic current frequency in rod bipolar cells, which was also dependent on dopaminergic activity, as it was abolished by dopamine type 1 receptor antagonism and under scotopic conditions. The effect was also abolished after 3-week negative lens-exposure but could be recovered using dopamine type 1 receptor agonism. Conclusions: Altogether, these results support a possible neuromodulatory role of glucagon in the retina of mammals as part of a dopaminergic activity-dependent synaptic pathway that is affected under myopia-inducing conditions.

The interplay between α7 nicotinic acetylcholine receptors, pannexin-1 channels and P2X7 receptors elicit exocytosis in chromaffin cells.

Pannexin-1 (Panx1) forms plasma membrane channels that allow the exchange of small molecules between the intracellular and extracellular compartments, and are involved in diverse physiological and pathological responses in the nervous system. However, the signaling mechanisms that induce their opening still remain elusive. Here, we propose a new mechanism for Panx1 channel activation through a functional crosstalk with the highly Ca2+ permeable α7 nicotinic acetylcholine receptor (nAChR). Consistent with this hypothesis, we found that activation of α7 nAChRs induces Panx1-mediated dye uptake and ATP release in the neuroblastoma cell line SH-SY5Y-α7. Using membrane permeant Ca2+ chelators, total internal reflection fluorescence microscopy in SH-SY5Y-α7 cells expressing a membrane-tethered GCAMP3, and Src kinase inhibitors, we further demonstrated that Panx1 channel opening depends on Ca2+ signals localized in submembrane areas, as well as on Src kinases. In turn, Panx1 channels amplify cytosolic Ca2+ signals induced by the activation of α7 nAChRs, by a mechanism that seems to involve ATP release and P2X7 receptor activation, as hydrolysis of extracellular ATP with apyrase or blockage of P2X7 receptors with oxidized ATP significantly reduces the α7 nAChR-Ca2+ signal. The physiological relevance of this crosstalk was also demonstrated in neuroendocrine chromaffin cells, wherein Panx1 channels and P2X7 receptors contribute to the exocytotic release of catecholamines triggered by α7 nAChRs, as measured by amperometry. Together these findings point to a functional coupling between α7 nAChRs, Panx1 channels and P2X7 receptors with physiological relevance in neurosecretion.

Cannabinoid Signaling Selectively Modulates GABAergic Inhibitory Input to OFF Bipolar Cells in Rat Retina.

Purpose: In the mammalian retina, cannabinoid type 1 receptors (CB1Rs) are well-positioned to alter inhibitory synaptic function from amacrine cells and, thus, might influence visual signal processing in the inner retina. However, it is not known if CB1R modulates amacrine cells feedback inhibition at retinal bipolar cell (BC) terminals. Methods: Using whole-cell voltage-clamp recordings, we examined the pharmacological effect of CB1R activation and inhibition on spontaneous inhibitory postsynaptic currents (sIPSCs) and glutamate-evoked IPSCs (gIPSCs) from identified OFF BCs in light-adapted rat retinal slices. Results: Activation of CB1R with WIN55212-2 selectively increased the frequency of GABAergic, but not glycinergic sIPSC in types 2, 3a, and 3b OFF BCs, and had no effect on inhibitory activity in type 4 OFF BCs. The increase in GABAergic activity was eliminated in axotomized BCs and can be suppressed by blocking CB1R with AM251 or GABAA and GABAρ receptors with SR-95531 and TPMPA, respectively. In all OFF BC types tested, a brief application of glutamate to the outer plexiform layer elicited gIPSCs comprising GABAergic and glycinergic components that were unaffected by CB1R activation. However, blocking CB1R selectively increased GABAergic gIPSCs, supporting a role for endocannabinoid signaling in the regulation of glutamate-evoked GABAergic inhibitory feedback to OFF BCs. Conclusions: CB1R activation shape types 2, 3a, and 3b OFF BC responses by selectively regulate GABAergic feedback inhibition at their axon terminals, thus cannabinoid signaling might play an important role in the fine-tuning of visual signal processing in the mammalian inner retina.

Physiological assessment of high glucose neurotoxicity in mouse and rat retinal explants.

One of the tissues of the central nervous system most affected by diabetes is the retina. Despite a growing understanding of the biochemical processes involved in glucose toxicity, little is known about the physiological consequences of chronic high glucose (HG) on individual neurons and neuronal circuits. Electroretinogram recordings suggest that retinal bipolar cells (BCs), which filter and transmit photoreceptor output to the inner retina, are among the first cells affected by diabetic conditions, and may therefore serve as sensitive early biomarkers for incipient neuronal damage caused in diabetes. Here, we comparatively assessed retinal integrity, calcium responses, and the electrophysiological profiles of specific BC types of mouse and rat organotypic retinal explants after 1 to 3 weeks in tissue culture, under moderate glucose (MG) and HG conditions. While the retinal layers of both rodent species displayed a progressively reduced thickness in culture, BCs retained their electrophysiological profiles and remained morphologically identifiable for up to 2 weeks. Responses to glutamate and endogenous inhibitory responses were routinely observed, indicating that the retinal circuitry remained intact during this period. Significant physiological differences between MG and HG conditions were evident in calcium signals and in the time course of responses to glutamate, but the voltage-gated current profiles of BCs displayed only minor variations. Overall, rat retina appeared slightly more sensitive to HG levels compared with mouse. In conclusion, electrophysiological analysis of neuronal function in rodent retinal explants is useful for the study of early damage due to HG neurotoxicity.

Publisher Correction: Electrical coupling between A17 cells enhances reciprocal inhibitory feedback to rod bipolar cells.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Electrical coupling between A17 cells enhances reciprocal inhibitory feedback to rod bipolar cells.

A17 amacrine cells are an important part of the scotopic pathway. Their synaptic varicosities receive glutamatergic inputs from rod bipolar cells (RBC) and release GABA onto the same RBC terminal, forming a reciprocal feedback that shapes RBC depolarization. Here, using patch-clamp recordings, we characterized electrical coupling between A17 cells of the rat retina and report the presence of strongly interconnected and non-coupled A17 cells. In coupled A17 cells, evoked currents preferentially flow out of the cell through GJs and cross-synchronization of presynaptic signals in a pair of A17 cells is correlated to their coupling degree. Moreover, we demonstrate that stimulation of one A17 cell can induce electrical and calcium transients in neighboring A17 cells, thus confirming a functional flow of information through electrical synapses in the A17 coupled network. Finally, blocking GJs caused a strong decrease in the amplitude of the inhibitory feedback onto RBCs. We therefore propose that electrical coupling between A17 cells enhances feedback onto RBCs by synchronizing and facilitating GABA release from inhibitory varicosities surrounding each RBC axon terminal. GJs between A17 cells are therefore critical in shaping the visual flow through the scotopic pathway.

On Biophysical Properties and Sensitivity to Gap Junction Blockers of Connexin 39 Hemichannels Expressed in HeLa Cells.

Although connexins (Cxs) are broadly expressed by cells of mammalian organisms, Cx39 has a very restricted pattern of expression and the biophysical properties of Cx39-based channels [hemichannels (HCs) and gap junction channels (GJCs)] remain largely unknown. Here, we used HeLa cells transfected with Cx39 (HeLa-Cx39 cells) in which intercellular electrical coupling was not detected, indicating the absence of GJCs. However, functional HCs were found on the surface of cells exposed to conditions known to increase the open probability of other Cx HCs (e.g., extracellular divalent cationic-free solution (DCFS), extracellular alkaline pH, mechanical stimulus and depolarization to positive membrane potentials). Cx39 HCs were blocked by some traditional Cx HC blockers, but not by others or a pannexin1 channel blocker. HeLa-Cx39 cells showed similar resting membrane potentials (RMPs) to those of parental cells, and exposure to DCFS reduced RMPs in Cx39 transfectants, but not in parental cells. Under these conditions, unitary events of ~75 pS were frequent in HeLa-Cx39 cells and absent in parental cells. Real-time cellular uptake experiments of dyes with different physicochemical features, as well as the application of a machine-learning approach revealed that Cx39 HCs are preferentially permeable to molecules characterized by six categories of descriptors, namely: (1) electronegativity, (2) ionization potential, (3) polarizability, (4) size and geometry, (5) topological flexibility and (6) valence. However, Cx39 HCs opened by mechanical stimulation or alkaline pH were impermeable to Ca2+. Molecular modeling of Cx39-based channels suggest that a constriction present at the intracellular portion of the para helix region co-localizes with an electronegative patch, imposing an energetic and steric barrier, which in the case of GJCs may hinder channel function. Results reported here demonstrate that Cx39 form HCs and add to our understanding of the functional roles of Cx39 HCs under physiological and pathological conditions in cells that express them.

Electrophysiological fingerprints of OFF bipolar cells in rat retina.

Retinal bipolar cells (BCs) divide photoreceptor output into different channels for the parallel extraction of temporal and chromatic stimulus properties. In rodents, five types of OFF BCs have been differentiated, based on morphological and functional criteria, but their electrophysiological characterization remains incomplete. This study analyzed OFF BCs with the patch clamp technique in acute slices of rat retina. Their specific voltage-dependent currents and glutamate responses are shown to represent individual fingerprints which define the signal processing and filtering properties of each cell type and allow their unequivocal identification. Two additions to the rat BC repertoire are presented: OFF BC-2', a variation of BC-2 with wider axonal arbours and prominent Na(+) currents, is described for the first time in rodents, and OFF BC-3b, previously identified in mouse, is electrophysiologically characterized in rat. Moreover, the glutamate responses of rat OFF BCs are shown to be differentially sensitive to AMPA- and kainate-receptor blockers and to modulation by nitric oxide (NO) through a cGMP-dependent mechanism. These results contribute to our understanding of the diversity and function of bipolar cells in mammals.

Acetylcholine induces GABA release onto rod bipolar cells through heteromeric nicotinic receptors expressed in A17 amacrine cells.

Acetylcholine (ACh) is a major retinal neurotransmitter that modulates visual processing through a large repertoire of cholinergic receptors expressed on different retinal cell types. ACh is released from starburst amacrine cells (SACs) under scotopic conditions, but its effects on cells of the rod pathway have not been investigated. Using whole-cell patch clamp recordings in slices of rat retina, we found that ACh application triggers GABA release onto rod bipolar (RB) cells. GABA was released from A17 amacrine cells and activated postsynaptic GABAA and GABAC receptors in RB cells. The sensitivity of ACh-induced currents to nicotinic ACh receptor (nAChR) antagonists (TMPH ~ mecamylamine > erysodine > DhßE > MLA) together with the differential potency of specific agonists to mimic ACh responses (cytisine >> RJR2403 ~ choline), suggest that A17 cells express heteromeric nAChRs containing the ß4 subunit. Activation of nAChRs induced GABA release after Ca(2+) accumulation in A17 cell dendrites and varicosities mediated by L-type voltage-gated calcium channels (VGCCs) and intracellular Ca(2+) stores. Inhibition of acetylcholinesterase depolarized A17 cells and increased spontaneous inhibitory postsynaptic currents in RB cells, indicating that endogenous ACh enhances GABAergic inhibition of RB cells. Moreover, injection of neostigmine or cytisine reduced the b-wave of the scotopic flash electroretinogram (ERG), suggesting that cholinergic modulation of GABA release controls RB cell activity in vivo. These results describe a novel regulatory mechanism of RB cell inhibition and complement our understanding of the neuromodulatory control of retinal signal processing.

Nitric oxide modulates the temporal properties of the glutamate response in type 4 OFF bipolar cells.

Nitric oxide (NO) is involved in retinal signal processing, but its cellular actions are only partly understood. An established source of retinal NO are NOACs, a group of nNOS-expressing amacrine cells which signal onto bipolar, other amacrine and ganglion cells in the inner plexiform layer. Here, we report that NO regulates glutamate responses in morphologically and electrophysiologically identified type 4 OFF cone bipolar cells through activation of the soluble guanylyl cyclase-cGMP-PKG pathway. The glutamate response of these cells consists of two components, a fast phasic current sensitive to kainate receptor agonists, and a secondary component with slow kinetics, inhibited by AMPA receptor antagonists. NO shortened the duration of the AMPA receptor-dependent component of the glutamate response, while the kainate receptor-dependent component remained unchanged. Application of 8-Br-cGMP mimicked this effect, while inhibition of soluble guanylate cyclase or protein kinase G prevented it, supporting a mechanism involving a cGMP signaling pathway. Notably, perfusion with a NOS-inhibitor prolonged the duration of the glutamate response, while the NO precursor L-arginine shortened it, in agreement with a modulation by endogenous NO. Furthermore, NO accelerated the response recovery during repeated stimulation of type 4 cone bipolar cells, suggesting that the temporal response properties of this OFF bipolar cell type are regulated by NO. These results reveal a novel cellular mechanism of NO signaling in the retina, and represent the first functional evidence of NO modulating OFF cone bipolar cells.

Nitric oxide signaling in the retina: what have we learned in two decades?

Two decades after its first detection in the retina, nitric oxide (NO) continues to puzzle visual neuroscientists. While its liberation by photoreceptors remains controversial, recent evidence supports three subtypes of amacrine cells as main sources of NO in the inner retina. NO synthesis was shown to depend on light stimulation, and mounting evidence suggests that NO is a regulator of visual adaptation at different signal processing levels. NO modulates light responses in all retinal neuron classes, and specific ion conductances are activated by NO in rods, cones, bipolar and ganglion cells. Light-dependent gap junction coupling in the inner and outer plexiform layers is also affected by NO. The vast majority of these effects were shown to be mediated by activation of the NO receptor soluble guanylate cyclase and resultant cGMP elevation. This review analyzes the current state of knowledge on physiological NO signaling in the retina.

The GABAergic system in the retina of neonate and adult Octodon degus, studied by immunohistochemistry and electroretinography.

UNLABELLED: In the vertebrate retina, gamma-aminobutyric acid (GABA) mediates inhibitory processes that shape the visual response and is also thought to have neurotrophic functions during retinal development. To investigate the role of GABAergic signaling at the beginning of visual experience, we used immunohistochemistry to compare the distribution of GABA, the two isoforms of glutamic acid decarboxylase GAD65/67, and the GABA receptor types A, B, and C, in neonate versus adult Octodon degus, a native South American rodent with diurnal-crepuscular activity and a high cone-to-rod ratio. In parallel, we used electroretinography to evaluate retinal functionality and to test the contribution of fast GABAergic transmission to light responses at both developmental stages. Neonate O. degus opened their eyes on postnatal day (P)0 and displayed an adult-like retinal morphology at this time. GABA, its biosynthetic sources, and receptors had a similar cellular distribution in neonates and adults, but labeling of the outer plexiform layer and of certain amacrine and ganglion cells was more conspicuous at P0. In neonates, retinal sensitivity was 10 times lower than in adults, responses to ultraviolet light could not be detected, and oscillatory potentials were reduced or absent. Blockade of GABA(A/C) receptors by bicuculline and TPMPA had no noticeable effect in neonates, while it significantly altered the electroretinogram response in adults. CONCLUSION: In spite of modest differences regarding retinal morphology and GABAergic expression, overall light response properties and GABAergic signaling are undeveloped in neonate O. degus compared to adults, suggesting that full retinal functionality requires a period of neural refinement under visual experience.